Computer Simulations of Enzymes

Enzymes are important catalysts in living systems, and understanding catalytic mechanisms of enzymes is an important task for modern biophysics and biochemistry. Computer simulations have emerged as very useful tools for understanding how enzymes work. In this dissertation, QM/MM MD simulations were applied to study the catalytic mechanisms of several enzymes, including sedolisin, S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases, and salicylic acid binding protein 2. For sedolisin, we focus on the acylation and deacylation reactions catalyzed by the enzymes. We proposed a general acid/base mechanism involving the Glu/Asp residues at the active site. MD and QM/MM free energy simulations on pro-kumamolisin show that the protonation of Asp164 would be able to trigger conformational changes and generate the functional active site for autocatalysis. The free energy simulations reported for SAMT, an AdoMet-dependent methyltransferase, showed that while the structure of the reactant complex containing salicylate, its natural substrate, is rather close to the corresponding TS structure, this is not the case for 4-hydroxybenzoate. The simulations demonstrated that additional energy is required to generate the TS-like structure for 4-hydroxybenzoate, consistent with the low activity of the enzyme toward this substrate. For protein lysine methyltransferase SET7/9, we showed that while the wild type SET7/9 may act like a mono-methylase, the Y245→A mutation could increase the ability of SET7/9 to add two more methyl groups on the target lysine. The substrate specificity of salicylic acid binding protein 2 (SABP2) has also been studied during my graduate study. This enzyme has promiscuous esterase activity toward a series of substrates, but shows high activity toward its natural substrate methyl salicylate (MeSA). We demonstrated that SABP2 seems to represent a case in which the enzyme itself might have not been perfectly evolved and that substrate-assisted catalysis (SAC) involving its natural substrate may be used to enhance the activity and achieve substrate discrimination. In addition to enzymes, the prediction of protein-protein interactions (PPI) is also included in my dissertation. We established a robust pipeline for PPI prediction by integrating multiple classifiers using random forests algorithm. This pipeline could be very useful for predicting PPI

Unexpected Reaction Pathway for Butyrylcholinesterase-Catalyzed Inactivation of Hunger Hormone Ghrelin

Extensive computational modeling and simulations have been carried out, in the present study, to uncover the fundamental reaction pathway for butyrylcholinesterase (BChE)-catalyzed hydrolysis of ghrelin, demonstrating that the acylation process of BChE-catalyzed hydrolysis of ghrelin follows an unprecedented single-step reaction pathway and the single-step acylation process is rate-determining. The free energy barrier (18.8 kcal/mol) calculated for the rate-determining step is reasonably close to the experimentally-derived free energy barrier (~19.4 kcal/mol), suggesting that the obtained mechanistic insights are reasonable. The single-step reaction pathway for the acylation is remarkably different from the well-known two-step acylation reaction pathway for numerous ester hydrolysis reactions catalyzed by a serine esterase. This is the first time demonstrating that a single-step reaction pathway is possible for an ester hydrolysis reaction catalyzed by a serine esterase and, therefore, one no longer can simply assume that the acylation process must follow the well-known two-step reaction pathway

Role of Histidine 547 of Human Dopamine Transporter in Molecular Interaction with HIV-1 Tat and Dopamine Uptake

HIV-1 Tat plays an important role in HIV-associated neurocognitive disorders (HAND) by disrupting neurotransmission including dopamine uptake by human dopamine transporter (hDAT). Previous studies have demonstrated that HIV-1 Tat directly binds to hDAT and some amino-acid mutations that attenuate the hDAT-Tat binding also significantly decreased dopamine uptake activity of hDAT. This combined computational-experimental study demonstrates that histidine-547 (H547) of hDAT plays a crucial role in the hDAT-Tat binding and dopamine uptake by hDAT, and that the H547A mutation can not only considerably attenuate Tat-induced inhibition of dopamine uptake, but also significantly increase the Vmax of hDAT for dopamine uptake. The finding of such an unusual hDAT mutant capable of both increasing the Vmax of hDAT for dopamine uptake and disrupting the hDAT-Tat binding may provide an exciting knowledge basis for development of novel concepts for therapeutic treatment of the HAND

QM/MM MD and Free Energy Simulations of G9a-Like Protein (GLP) and Its Mutants: Understanding the Factors that Determine the Product Specificity

Certain lysine residues on histone tails could be methylated by protein lysine methyltransferases (PKMTs) using S-adenosyl-L-methionine (AdoMet) as the methyl donor. Since the methylation states of the target lysines play a fundamental role in the regulation of chromatin structure and gene expression, it is important to study the property of PKMTs that allows a specific number of methyl groups (one, two or three) to be added (termed as product specificity). It has been shown that the product specificity of PKMTs may be controlled in part by the existence of specific residues at the active site. One of the best examples is a Phe/Tyr switch found in many PKMTs. Here quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations are performed on wild type G9a-like protein (GLP) and its F1209Y and Y1124F mutants for understanding the energetic origin of the product specificity and the reasons for the change of product specificity as a result of single-residue mutations at the Phe/Tyr switch as well as other positions. The free energy barriers of the methyl transfer processes calculated from our simulations are consistent with experimental data, supporting the suggestion that the relative free energy barriers may determine, at least in part, the product specificity of PKMTs. The changes of the free energy barriers as a result of the mutations are also discussed based on the structural information obtained from the simulations. The results suggest that the space and active-site interactions around the ε-amino group of the target lysine available for methyl addition appear to among the key structural factors in controlling the product specificity and activity of PKMTs

MD results for the F1209Y mutant.

<p>(A) A representative active-site structure along with the average values of some structural parameters of the reactant complex for the first methyl transfer. (B) Left: the two-dimensional plot of <i>r</i>(C_M…N_ζ) and <i>θ</i> distributions of the reactant complex for the first methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C_M…N_ξ) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (C) The structure of the reactant complex for the second methyl transfer. (D) Left: the two-dimensional plot of <i>r</i>(C_M…N_ζ) and <i>θ</i> distributions of the reactant complex for the second methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C_M…N_ξ) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (E) The structure near the transition state for the first methyl transfer. (F) The structure near the transition state for the second methyl transfer.</p

MD results for the Y1124F mutant.

<p>(A) A representative active-site structure along with the average values of some structural parameters of the reactant complex for the first methyl transfer. (B) Left: the two-dimensional plot of <i>r</i>(C_M…N_ζ) and <i>θ</i> distributions of the reactant complex for the first methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C_M…N_ξ) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (C) The structure of the reactant complex for the second methyl transfer. (D) Left: the two-dimensional plot of <i>r</i>(C_M…N_ζ) and <i>θ</i> distributions of the reactant complex for the second methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C_M…N_ξ) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (E) The structure of the reactant complex for the third methyl transfer. (F) Left: the two-dimensional plot of <i>r</i>(C_M…N_ζ) and <i>θ</i> distributions of the reactant complex for the third methyl transfer; Middle: the free-energy change as a function of <i>r</i>(C_M…N_ξ) obtained from the distributions; Right: the free-energy change as a function of <i>θ</i> obtained from the distributions. (G) A representative active-site structure along with the average values of some structural parameters near the transition state for the first methyl transfer. (H) The structure near the transition state for the second methyl transfer. (I) The structure near the transition state for the third methyl transfer.</p

Free energy profiles of methyl transfer processes in the Y1124F mutant.

<p>The first methyl transfer: blue and solid line with a free energy barrier of 13.9 kcal/mol; the second methyl transfer: red and dashed line with a free energy barrier of 15.9 kcal/mol; the third methyl transfer: green and dashed line with a free energy barrier of 13.3 kcal/mol.</p

QM/MM free energy simulations of the reaction catalysed by (<i>4S</i>)-limonene synthase involving linalyl diphosphate (LPP) substrate

<p>A large number of terpenoid natural products are known to exist in nature. Terpene synthases are pivotal enzymes for the biosynthesis of diverse terpenoid skelotons. Monoterpene synthases are one type of terpene synthases responsible for the production of several hundreds of natural monoterpenes based on a very limited pool of substrates. Therefore, understanding detailed catalytic mechanisms of those enzymes are important for understanding the product specificity of terpene synthases. In this study, we present a detailed mechanistic description of the biosynthesis of the (<i>4S</i>)-α-terpinyl carbocation from (<i>3S</i>)-linalyl diphosphate (LPP) catalysed by (<i>4S</i>)-limonene synthase (LS) using two-dimensional QM/MM free energy (2D-PMF) simulations. Our estimated free energy barrier is in a reasonable agreement with the corresponding experimental kinetic data. We also perform the one-dimensional QM/MM free energy (1D-PMF) simulations and show that His579 can act as a general base to deprotonate (<i>4S</i>)-α-terpinyl carbocation and to generate the limonene product.</p